Factors that Influence Subcellular Mitochondrial Positioning

Miro1 is an essential mitochondrial adaptor protein that supports strategic mitochondrial positioning to specific sites within the cell. The subcellular location of mitochondria is important for localized energy and metabolite production and signaling from mitochondria. The factors that regulate mitochondrial positioning and the way in which Miro1 responds to these signals to coordinate mitochondrial dynamics is unknown. Previous research has demonstrated that reactive oxygen species (ROS) influence mitochondrial motility; therefore, we chose to investigate the effect of different sources of ROS on Miro1 and mitochondrial networking. With a gene-edited cell line expressing GFP-tagged Miro1, we performed immunofluorescence assays with laser scanning confocal microscopy, immunoprecipitation, and mass spectrometry to quantify the effects of ROS generated in different cellular compartments on Miro1 protein binding partners, Miro1 oxidation and changes in mitochondrial structure. We found that the location of ROS production had very different effects on Miro1 association with the mitochondria, mitochondrial networking, and mitochondrial motility. We also generated preliminary data showing that Miro1 contains cysteines capable of being oxidized. This project has revealed that different sources of ROS induce modifications to the Miro1 protein and diferentially coordinates mitochondrial positioning and networking. Since disrupted mitochondrial dynamics are central to many disease states, including neurodegeneration and tumor metastasis, these results may yield valuable insight into the role Miro1 plays in the establishment of these ROS-associated diseases and Miro1’s potential as a drug target